Project Summary A variety of cellular processes are commonly subverted to encourage the proliferation of cancer cells, one of which is the unfolded protein response (UPR) that occurs in the endoplasmic reticulum (ER). Importantly, there is also a strong connection between UPR and inflammation or neuronal health. Many neurodegenerative diseases and diseases of aging have connections to the UPR. We have recently discovered a new form of BiP regulation, AMPylation by the protein Fic. We observe that Fic adds an adenosine monophosphate (AMP) molecule to a threonine near the ATP binding site of BiP during normal growth conditions. This modification rapidly is removed by the same enzyme Fic under multiple ER stress-inducing conditions. We recently have shown that the regulation of BiP by Fic is essential for maintaining neuronal homeostasis. Since our discovery of Fic domains that mediate AMPylation, other diverse activities performed by bacterial Fic domains have been identified. These studies have revealed the molecular plasticity of Fic domains in its ability to utilize diverse substrates. Despite these studies, there are many different Fic proteins that remain to be characterized, both in catalytic activity, biological function, and molecular targets. We propose three projects that will further our understanding of the biology of Fic enzymes and the chemistry they use, both in vitro and in vivo. First, when the ER is stressed, Fic changes from an AMPylator to a deAMPylator, and the key to this regulation is breaking a salt bridge in Fic's active site. We want to understand the biochemistry regulating this switch. Second, our studies with Drosophila genetics show that Fic is required for neuronal plasticity. We are therefore interested in understanding what role Fic plays in mammalian biology using transgenic mice. Third, there are many different Fic proteins that remain to be characterized, both in catalytic activity, biological function, and molecular targets. We plan to investigate these orphan Fic domains and identify their biological activities and substrates.